Md. Nazmul Hasan, Guo Renkai, Mayumi Yoshida, Hiroto Ohta, JAE MAN LEE, takahiro kusakabe, Akinori Hirashima, Screening for inhibitor of recombinant Drosophila melanogaster tyramine-β-hydroxylase, Medicinal chemistry and computer aided drug design, 2015.11, Biogenic amines are common biologically active substances extended within the whole animal kingdom where they play vital roles as signal transducer as well as regulator of cell functions. One of these biogenic amines called octopamine (OA) originated in the insect nervous system is synthesized from tyramine (TA) by the catalysis of tyramine-β-hydroxylase (TβH). Both tyramine and octopamine act as neurotransmitters, neurohormones and neuromodulators in the arthropod nervous system. Herein, inhibitory activity of 1-arylimidazole-2(3H)-thiones (AITs) were tested on Drosophila tyramine-β-hydroxylase (DmTβH) purified from recombinant Bombyx mori strain. Radiolabelled 3H-TA was used to analyze the activity of inhibitors and the radioactivity was measured by liquid scintillation counter. Different concentrations of AITs exhibited inhibitory effects on DmTβH which was ID50 values ranging from 0.020 nM to 2.511 µM. The most effective compounds for DmTβH inhibition were 2-Me AIT; 2-Et AIT; 4-CF3 AIT; 2,4-Me2 AIT; 2,6-Me2 AIT; 2,3,4-Cl3 AIT; 3,5-(MeO)2 AIT; 2-Me,4-Cl AIT; 2-Cl,5-Me AIT; 2-MeO,4-NO2 AIT; unsubstituted AIT; 4-MeO AIT; 4-MeS AIT; 2,5-(MeO)2 AIT and 2-Me,6-Cl AIT with ID50 values 5.820, 1.887, 0.235, 1.246, 0.020, 0.301, 3.175, 0.154, 0.796, 1.201 nM and 0.004, 0.009, 0.008, 0.009, 0.003 µM respectively. DmTβH was inhibited as a dose dependent manner at pH 7.6 and 25 ºC during a 30 min of incubation. The inhibitory activity of AITs on DmTβH may be considered as lead compounds to control insect population after further extensive studies..

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【Purpose】The aim is to express and characterize the DmTbH for further prospective pharmacological studies.【Methods and Results】DmTbH was isolated and followed by generation of expression vector for construction of recombinant virus. After injection of the bacmid into the 5th instar silkworm larvae, the recombinant protein was purified from the haemolymph. Finally, the TbH assay was performed by radiochemical method. SDS-PAGE demonstrated a single 70KDa band in haemolymph of purified recombinant protein. To determine the expression level of DmTBH, the western blot analysis was conducted. The products formed by the incubation of enzyme reaction mixture were separated and detected by the reverse phase HPLC. The optimum pH, temperature and incubation time that allow tyramine to convert into octopamine were 7.6, 25 °C and 30 min, respectively..